End stage liver disease, a consequence common to various hepatic diseases, both acute and chronic, continues to be a major cause of morbidity and mortality worldwide. In fact, chronic liver disease and cirrhosis, are the 12th leading cause of death in the US. Liver transplantation is the only effective available treatment, which is plagued by a dearth of available organs. Continued research is focusing on innovative surgical techniques, cell therapy, stem cell differentiation, artificial liver devices and generation of organs de novo. Broadly defined as Hepatic Regenerative Medicine, this field has gained momentum with the advent of induced pluripotential stem cells, liver-on-chip including the use of micro-physiological systems (MPS) and the use of decellularized liver matrices for differentiation. However, the relative success of these modalities has been limited, mandating improved and thorough understanding of liver physiology for application to these technologies. Continued explication of the complex cell-molecule circuitry for the most relevant pathways in hepatic physiology will be pertinent to the continued success of hepatic regenerative medicine, which may yield novel ways to treat patients with acute liver failure, toxicant- and drug- induced injury. Improved understanding of signaling pathways enabling LR will also have applications in improving liver transplantation outcomes, treatment of complications like small-for-size syndrome and accelerating post-transplantation recovery in both recipients and living donors. One major master regulator of hepatocyte biology that others and we have contributed in identifying, is the Wnt/b-catenin pathway. We have helped identify its key roles in hepatic growth and development, metabolic zonation and liver regeneration (LR). The impetus behind requesting continuation of our study are important observations made over the last 4 years. Liver-specific b-catenin- KO or b-Cat-LKO mice lack pericentral gene expression, thus liver zonation (LZ) is a key function of b-catenin. Likewise, liver regeneration (LR) after partial hepatectomy (PH) depends on hepatocyte-b-catenin, which regulates cyclin-D1 expression at 24h, & in turn induces proliferation at 40h. b-Catenin, for both LZ and LR, is under the control of Wnt since LRP5-6-LKO mice lacking these Wnt co-receptors, phenocopy b-Cat-LKO. To address cell source of Wnt, Wntless (Wls), critical for Wnt secretion, was deleted by breeding Wls-floxed mice to cell-specific cre lines: LysM-cre (macrophages or Mac), Alb-cre (liver epithelial cells). Lrat-cre (hepatic stellate cells or HSC) and Lyve1-cre (endothelial cells or EC). Only EC-Wls-KO phenocopied b-Cat-LKO and LRP5-6-LKO in both loss of LZ and delay in LR after PH. Using microdissection and cell fractionation, Wnt2 and Wnt9b, from central vein (CV) EC and sinusoidal EC (SEC) were shown to be important for LZ and during LR, respectively. The current proposal has three specific aims. Aim 1 will determine effect of conditional loss of Wnt2 and Wnt9b from EC by interbreeding Wnt2-9b-double floxed mice, Rosa-stop-floxed-EYFP mice and Lyve1- cre. The EC-Wnt2-9b-KO-EYFP mice will be characterized for LZ at baseline, and for LR after PH. In Aim 2, based on preliminary observations of highest Fzd7 expression in zone 3 hepatocytes and increase in Fzd7 expression after PH, we will use Fzd7-floxed mice and Alb-cre mice or i.p. injection of AAV8-TBG-Cre, to eliminate Fzd7 from hepatocytes for characterization of LZ and LR. Additionally, using state-of-the-art and innovative Fzd7-agonist, we will investigate its effect on LR after PH in a living donor-like scenario in mice. At the same time, we will test effect of overexpression of Fzd7 using AAV8-TBG-Fzd7 on LR after PH. In aim 3, we will investigate the upstream effectors that regulate Wnt2/9b expression in CVEC at baseline and in SEC after PH during LR. Based on preliminary observations, we posit persistent hypoxia in CVEC and shear stress on SEC as the most relevant upstream effectors of Wnt2 & 9b in regulating LZ and LR respectively. We propose to address these processes in vitro in microfluidic devices and in vivo using hypoxia chambers and pharmacological modulators of hepatic blood flow after PH. Thus, our central hypothesis is that specific biophysical processes induce Wnt2/9b expression in EC to activate b-catenin at baseline in PC hepatocytes as part of LZ and spatiotemporally in midzone and periportal hepatocytes during LR, via the Fzd7 receptor in a paracrine manner.